The present invention relates to domestic fowl eggs having a high content of arachidonic acid and optionally docosahexaenoic acid, and a process for production thereof from egg-laying domestic fowls raised by feeding xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid, as well as lipid having a high content of arachidonic acid and optionally docosahexaenoic acid, obtained from said domestic fowl eggs, a process for production thereof, and their use.
Although eggs having a high degree of nutritional value and are excellent foods that contain a good balance of nutrients, due to their high cholesterol content, their consumption must be restricted. In recent years, research has therefore been conducted to introduce various drugs into eggs to inhibit increases in blood cholesterol levels, and numerous research has been reported.
On the other hand, after it was reported that xcfx893 highly unsaturated fatty acids contained in fish oil have an effect of lowering blood cholesterol (Japanese Unexamined Patent Publication No. 54-154533), a technology was developed in which large amounts of docosahexaenoic acid and eicosapentaenoic acid are introduced into the egg yolks of domestic fowl eggs by raising egg-laying domestic fowls on feed to which was added fish oil containing xcfx893 highly unsaturated fatty acids (Japanese Examined Patent Publication No. 3-36493).
However, reports of substances having the effect of lowering blood cholesterol were not limited to xcfx893 highly unsaturated fatty acid. This effect is also observed in xcfx896 highly unsaturated fatty acids such as xcex3-linolenic acid (Agric. Biol. Chem., 50, 2483-2491 (1986)), dihomo-xcex3-linolenic acid and arachidonic acid (xe2x80x9cLipid Metabolism of Liver Diseasexe2x80x9d, Chugai Medical Co., Ltd., 1994, pp. 127-130). However, a method of for increasing amounts of xcfx896 highly unsaturated fatty acids, and particularly arachidonic acid, in eggs has not yet been developed. Moreover, in recent years, questions have been raised regarding the ratio of xcfx896 highly unsaturated fatty acids and xcfx893 highly unsaturated fatty acids. Consequently, instead of the conventional technology for increasing amount of eicosapentaenoic acid or docosahexaenoic acid in egg yolks, there has been a strong desire for a method of containing xcfx896 highly unsaturated fatty acid and xcfx893 highly unsaturated fatty acid in egg yolks in a favorable balance.
In view of these circumstances, it is stated for the amounts of ingested fatty acids in the section concerning ingestion of fatty acids in the Nutritional Requirements of the Japanese (5th Revision) that, xe2x80x9cConsistent opinions have not yet been obtained regarding the ratio of ingestion of n-6 (xcfx896) fatty acids and n-3 (xcfx893) fatty acids. The results of a nutritional survey of Japanese showed that many of those surveyed fatty acids are ingested in the ratio of roughly 4:1, and this ratio is considered to be suitable at the present stage.xe2x80x9d (Ministry of Health and Welfare, Health Service Bureau, Health Promotion and Nutrition Dept. ed.: xe2x80x9cNutritional Requirements of the Japanese, 5th Revisionxe2x80x9d, 1st edition, 1994, pp. 56-58).
In addition, it has also been recently reported that arachidonic acid and docosahexaenoic acid are contained in mother""s milk, and that they are useful in the growth and development of infants (xe2x80x9cAdvances in Polyunsaturated Fatty Acid Researchxe2x80x9d, Elsevier Science Publishers, 1993, pp 261-264). Moreover, their importance has also been reported in the height and brain development of the fetus (Proc. Natl. Acad. Sci. USA, 90, 1073-1077 (1993), Lancet, 344, 1319-1322 (1994)).
With this in mind, attempts were made to add arachidonic acid and docosahexaenoic acid, for which there are large differences in the fatty acid composition between mother""s milk and infant formula, to infant formula. Although infant formula containing fish oil is currently available on the market for the purpose of adding docosahexaenoic acid to a infant formula, eicosapentaenoic acid contained in fish oil is inherently hardly contained at all in mother""s milk. According to the results of recent research, this substance is not always favorable for the growth and development of premature infants (xe2x80x9cAdvances in Polyunsaturated Fatty Acid Researchxe2x80x9d, Elsevier Science Publishers, 1993, pp. 261-264). U.S. Pat. No. 5,374,657 describes an invention relating to an oil to be added to milk product for infants, which oil blend comprises an edible oil in microbial cells containing docosahexaenoic acid and an edible oil in microbial cells containing arachidonic acid, but a small amount of eicosapentaenoic acid. However, since this involves direct administration of microbiological oil to premature infants and nursing infants, considerable cautions are required in terms of safety.
On the other hand, U.S. Pat. No. 4,670,285 discloses an amount of fatty acids such as arachidonic acid required by infants, and a blend of egg yolk oil and vegetable oil as an edible fat product for incorporation into an infant formula for providing these fatty acids. The egg yolk lipid used here can be said to have a lower amount of eicosapentaenoic acid and be an extremely safe raw material in comparison with the above-mentioned fish oil and microbial oil. However, since this egg yolk lipid contains only small amounts of arachidonic acid and docosahexaenoic acid (roughly 1.5% arachidonic acid and roughly 1.7% docosahexaenoic acid per the total fatty acids in the egg yolk lipid), it is uneconomical.
xcfx893 highly unsaturated fatty acids have an abundant supply source, namely fish oil which have no problems whatsoever in terms of safety. However, there are surprisingly few supply sources of xcfx896 highly unsaturated fatty acids, such as arachidonic acid. For example, although it is known that large amount of arachidonic acid are contained in liver, there are few opportunities for its consumption as a food. In addition, it is present only in small amounts in other meats (roasts or filet portions).
Therefore, extensive efforts were made to seek a supply source of xcfx896 highly unsaturated fatty acids such as arachidonic acid in the microbial world. A technology was developed relating to the production of xcfx896 highly unsaturated fatty acids by microorganisms represented by molds belonging to genus Mortierella, subgenus Mortierella (Japanese Unexamined Patent Publication No. 63-044891). However, although this technology was considered to have a high degree of safety, it did not expand significantly due to the problem of being of microbial origin.
Thus, there was a strong desire for the development of domestic fowl eggs fortified with arachidonic acid and optionally docosahexaenoic acid, as well as a lipid having a high content of arachidonic acid, and optionally docosahexaenoic acid extracted from those domestic fowl eggs for use as safe sources of arachidonic acid.
Thus, the present invention provides domestic fowl eggs having a high content of arachidonic acid and optionally docosahexaenoic acid, and a process for production thereof, as well as a lipid having a high content of arachidonic acid and optionally docosahexaenoic acid obtained from said domestic fowl eggs, a process for production thereof and its use.
As a result of various research to achieve the above-mentioned object, the inventors of the present invention found that domestic fowl eggs obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acids and optionally xcfx893 highly unsaturated fatty acids have a high contents of arachidonic acid and optionally docosahexaenoic acid, that are at least 2%, respectively, of the total amount of fatty acids in the egg yolk, thereby leading to completion of the present invention.
Thus, the present invention provides domestic fowl eggs having a high content of arachidonic acid and optionally docosahexaenoic acid obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid.
Moreover, the present invention provides a production process of domestic fowl eggs having a high content of arachidonic acid and optionally docosahexaenoic acid comprising feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid.
In addition, the present invention provides a lipid having a high content of arachidonic acid and optionally docosahexaenoic acid obtained from domestic fowl eggs obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid.
Moreover, the present invention provides a production process of a lipid having a high content of arachidonic acid and optionally docosahexaenoic acid comprising extracting lipid having a high content of arachidonic acid and optionally docosahexaenoic acid from domestic fowl eggs obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid.
Moreover, the present invention provides a food having arachidonic acid and optionally docosahexaenoic acid, containing at least 0.001% by weight of a lipid having a high content of arachidonic acid and optionally docosahexaenoic acid obtained by extracting from domestic fowl eggs obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid.
In a preferable embodiment of the above-mentioned invention, the xcfx896 highly unsaturated fatty acid is at least one of the fatty acids selected from the group consisting of xcex3-linolenic acid, dihomo-xcex3-linolenic acid and arachidonic acid. This is preferably used in at least one of the forms selected from the group consisting of free fatty acid, salt, ester, triacylglycerol, diacylglycerol, monoacylglycerol, glycerophospholipid, glyceroglycolipid, sphingophospholipid and sphingoglycolipid.
Preferably, xcfx896 highly unsaturated fatty acid is given to domestic fowls either alone or as a mixture in the form of an oil or extract residue obtained by extracting from dried or wet microbial cells of a microorganism having the ability to produce arachidonic acid, or dried or wet microbial cells of a microorganism having the ability to produce arachidonic acid.
Microorganisms having the ability to produce arachidonic acid are preferably of the genus Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium and Saprolegnia.
Preferably, xcfx893 highly unsaturated fatty acid is at least one of the fatty acids selected from the group consisting of xcex1-linolenic acid, 8,11,14,17-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, 7,10,13,16,19-docosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid. These are preferably used in at least one of the forms selected from the group consisting of free fatty acid, salt, ester, triacylglycerol, diacylglycerol, monoacylglycerol, glyerophospholipid, glyceroglycolipid, sphingophospholipid and sphingoglycolipid.
Preferably, xcfx893 highly unsaturated fatty acid is given to domestic fowls either alone or as a mixture in the form of fish oil, fish powder, fish refuse, fish oil extract, an oil or extract residue obtained by extracting from dried or wet microbial cells of a microorganism having the ability to produce docosahexaenoic acid, or dried or wet microbial cells of a microorganism having the ability to produce docosahexaenoic acid.
Microorganisms having the ability to produce docosahexanoic acid are preferably of the genus Crypthecodimium, Isochrysis, Nanochloropsis, Chaetoceros, Phaeodactylum, Amphidinium, Gonyaulax, Peridimium, Chroomonas, Cryptomonas, Hemiselmis, Chilomonas, Chlorella, Histiobranchus, Coryphaenoides, Thraustchytrium, Schizochytrium Conidiobolus and Entomorphthora.
The present invention was completed on the basis of the above findings. Namely, the present invention relates to domestic fowl eggs having a high content of arachidonic acid and optionally docosahexaenoic acid, and a process for production thereof comprising feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid, as well as lipid having a high content of arachidonic acid and optionally docosahexaenoic acid obtained by extracting from the resulting domestic fowl eggs, and particularly the yolks, a process for production thereof, and the use thereof.
The egg-laying domestic fowls used in the present invention may be any of the large number of types of fowl referred to as poultry provided their eggs are edible, examples of which include chickens, quail, ducks and crossbreeds of wild and domestic ducks.
The xcfx896 highly unsaturated fatty acid of the present invention is that having at least 18 carbon atoms, preferably at least 20 carbon atoms, and at least 3 double bonds which start from the 6th carbon atom counting from the carbon atom on the methyl group terminal of the fatty acid, examples of which include xcex3-linolenic acid, dihomo-xcex3-linolenic acid and arachidonic acid. Although these fatty acids can be used either alone or as a mixture, it is preferable that they include at least arachidonic acid. In addition, these fatty acids can be added in various forms. Examples of these forms include salts, atoxic metal salts, for example, alkaline metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts, esters such as methylesters, ethylesters and propylesters, triacylglycerols, diacylglycerols, monoacylglycerols, glyerophospholipids, glyceroglycolipids, sphingophospholipids and sphingoglycolipids.
The xcfx896 highly unsaturated fatty acids of the present invention may be chemically synthesized, or derived from an animal, plant or microorganism and so forth that contains xcfx896 highly unsaturated fatty acid, or that which is isolated, extracted, purified or is a residue thereof, an example of which is an oil or extract residue obtained from a microorganism having an ability to produce arachidonic acid. In addition, it is desirable that the oil containing xcfx896 highly unsaturated fatty acid of the present invention contains at least 5.5%, preferably at least 10%, and more preferably at least 20% arachidonic acid with respect to total fatty acids.
Moreover, in the present invention, the xcfx896 highly unsaturated fatty acid can be used in combination with other fatty acids such as linoleic acid, xcfx893 highly unsaturated fatty acids and so forth.
The xcfx893 highly unsaturated fatty acid has at least 18 carbon atoms and at least 2 double bonds which start from the 3rd carbon atom counting from the carbon atom of the methyl group terminal of the fatty acid. Examples of the xcfx893 highly unsaturated fatty acid used in the present invention are xcex1-linolenic acid, 8,11,14,17-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, 7,10,13,16,19-docosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid. For the sake of brevity, the double bond locations will be omitted. In addition, these fatty acids can be added in various forms.
Examples of forms in which the above-mentioned fatty acids can be added include salts, atoxic metal salts, for example, alkaline metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts, esters such as methylesters, ethylesters and propylesters, triacylglycerols, diacylglycerols, monoacylglycerols, glycerophospholipids, glyceroglycolipids, sphingophospholipids and sphingoglycolipids.
The xcfx893 highly unsaturated fatty acids of the present invention may be chemically synthesized or derived from an animal, plant or microorganism and so forth that contains xcfx893 highly unsaturated fatty acid, or that which is isolated, extracted or purified therefrom, examples of which include fish oil, fish powder, fish refuse, fish oil extract, an oil or extract residue obtained from microorganisms having an ability to produce docosahexanoic acid and so forth.
Typical examples of fish oils include sardine oil, herring oil, tuna oil, bonito oil, saury oil and menherden oil. Although there are no particular limitations on the type of fish oil used in the present invention, since the fatty acid composition in the oil varies according to the type of fish, selecting and using a fish oil having a high content of docosahexaenoic acid and a low content of eicosapentaenoic acid is preferable for obtaining eggs having a low content of eicosapentaenoic acid.
In the present invention, all microorganisms can be used provided they have an ability to produce arachidonic acid or docosahexaenoic acid. Examples of microorganisms having an ability to produce arachidonic acid include those of the genus Mortierella, Conidiobolus, Phythium, Phytophthora, Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium and Saprolegnia.
In the case of the genus Mortierella, microorganisms belonging to the genus Mortierella, subgenus Mortierella can be used, examples of which include Mortierella elongata IFO 8570, Mortierella exiqua IFO 8571, Mortierella hygrophila IFO 5941 and Mortierella alpina IFO 8568. All of these strains can be acquired without restriction from the Institute for Fermentation Osaka. In addition, the present inventions can also use Mortierella elongata SAM 0219 (FERM P-8703) (FERM BP-1239).
Mortierella alpina is particularly preferable in the present invention because it intracellularly accumulates a large amount of arachidonic acid. In addition, since this microorganism produces hardly eicosapentaenoic acid at normal temperatures (preferably 20 to 30xc2x0 C.), it is suited extremely well for obtaining eggs having a low content of eicosapentaenoic acid. Examples of Mortierella alpina other than that listed above include Mortierella alpina ATCC 16266, ATCC 42430, ATCC 32221, CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70 and CBS754.68.
Examples of microorganisms that have an ability to produce docosahexaenoic acid include members of the phylum Chromophycota, such as of the genus Crypthecodimium, Isochrysis, Nanochloropsis, Chaetoceros, Phaeodactylum, Amphidinium, Gonyaulax, Peridimium, Chroomonas, Cryptomonas, Hemiselmis, Chilomonas, as well as members of the phylum of Chlorophycota such as of the genus Chlorella, Histiobranchus, Coryphaenoides, Thraustchytrium, Schizochytrium Conidiobolus and Entomorphthora. Examples of Crypthecodimium include Crypthecodimium cohnii ATCC 30021, while examples of Thraustchytrium include Thraustchytrium aureum ATCC 34304. These strains can be acquired without restriction from the American Type Culture Collection.
Microorganisms having an ability to produce arachidonic acid or microorganisms having an ability to produce docosahexaenoic acid can be cultured in accordance with routine methods. For example, the spores, mycelia or pre-culture liquid obtained by culturing in advance of said microorganism can be cultured by inoculating into liquid medium or solid medium. An oil containing highly unsaturated fatty acid such as arachidonic acid or docosahexaenoic acid is intracellularly accumulated as a result of culturing.
Following completion of culturing, cultured microbial cells are obtained from the culture by commonly employed solid-liquid separation techniques such as centrifugal separation and filtration. Dry microbial cells are obtained by washing the cultured microbial cells with an ample amount of water and then drying. Drying can be performed by freeze-drying, spray drying and so forth. The dried microbial cells are preferably extracted with organic solvent in the presence of a nitrogen gas. Examples of organic solvents that can be used include ethyl ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether and acetone. In addition, favorable results can also be obtained by alternating extraction with methanol and petroleum ether or by extraction using a single-layer solvent of chloroform, methanol and water. An oil can then be obtained containing highly unsaturated fatty acid such as arachidonic acid or docosahexaenoic acid by distilling off the organic solvent from the extract under a reduced pressure.
According to the present invention, in the case of feeding domestic fowls with oil derived from microorganisms having an ability to produce arachidonic acid or oil derived from microorganisms having an ability to produce docosahexaenoic acid, in addition to the oil extracted from cultured microbial cells, the oil can be fed in forms including culture liquid taken during the course of culturing or that sterilized culture liquid, culture liquid taken at completion of culturing or that sterilized culture liquid, cultured microbial cells collected from those respective culture liquids or those dried microbial cells, or residue obtained after extracting oil from the microbial cells.
There are no particular limitations on the manner in which domestic fowls are fed with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid. For example, these highly unsaturated fatty acids can be added to feed, or given in drinking water followed by emulsification using O/W emulsifier, to a concentration of 0.1-3% by weight (Japanese Unexamined Patent Publication No. 60-105471) or subcutaneously administered (Japanese Unexamined Patent Publication No. 5-292583). In addition, in order to produce the domestic fowl eggs of the present invention, it is preferable to feed said domestic fowls witht the fatty acids at a dose of at least 100 mg/day/fowl, and preferably at least 400 mg/day/fowl. Although merely adding xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid in feed is the simplest method, since its oxidation cannot be avoided, there are problems in terms of its quality control as well as the generation of a foul odor in the vicinity of the breeding area.
However, in the case of using microorganisms that have an ability to produce arachidonic acid or microorganisms that have an ability to produce docosahexaenoic acid, although problems similar to those above occur in the case of adding its extracted oil to feed, in the case of using the microbial cells of these microorganisms, the oil in the microbial cells is stable and, there are immeasurable advantages in terms of costs, since the extraction procedure can be omitted, as well as in terms of being able to provide other nutrients (proteins, sugars and so forth).
With the exception of trace amounts contained in the egg white and egg shell, virtually all of the lipid in domestic fowl eggs is contained in the yolk, and the majority of this egg yolk lipid is said to be bound to protein. Thus, in the case of extracting lipid from yolk, the amount and composition of the extracted lipid varies according to the type of solvent used and extraction conditions. If the egg yolk is treated in advance using enzyme (protease preparation containing lipase), an amount of lipid extracted by solvent extraction increases. Examples of typically used effective extraction agents include a mixture of ethanol and ether (3:1) and a mixture of chloroform and methanol (1:1), and these solvents are suited for extraction of all yolk lipids. In addition, by first freeze-drying the yolk in advance to form a powder, and then extracting with a mixture of chloroform and methanol, lipid is completely extracted. In particular, ethanol or hexane alone, a mixture of ethanol and hexane or a mixture of ethanol and water are preferable since they are solvents suitable for use in foods.
Fatty acid analysis can be performed in accordance with routine methods. For example, fatty acids can be measured by gas chromatography, high-performance liquid chromatography and so forth.
The egg yolk lipid of the present invention thus obtained is richer in arachidonic acid than conventional eggs. More specifically, this egg yolk lipid contains at least 2%, preferably at least 2.7%, and more preferably at least 3% arachidonic acid with respect to the total fatty acids contained in the egg yolk. In addition, in the present invention, fatty acid analysis of the resulting lipid having a high content of arachidonic acid and docosahexaenoic acid indicates a ratio of 1 to 12 parts by weight of docosahexaenoic acid to 1 to 12 parts by weight of arachidonic acid. Moreover, said egg yolk lipid is characterized by demonstrating a ratio of at least 5 parts by weight of arachidonic acid to 1 part by weight of eicosapentaenoic acid.
Therefore, the lipid of the present invention having a high content of arachidonic acid and optionally docosahexaenoic acid extracted from domestic fowl eggs, and particularly the egg yolks, obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid has a low ratio of eicosahexaenoic acid with respect to total fatty acids in the egg yolk even in the case the above-mentioned domestic fowls are raised using fish oil for the xcfx893 highly unsaturated fatty acid. Thus, an extracted lipid is obtained that has high ratios of arachidonic acid and optionally docosahexaenoic acid, which can be effectively used in formula for premature infants, infant formula, a follow-up formula or as a milk product for expectance or nursing mother. Namely, a powdered formula that is closer to natural mother""s milk can be obtained by adding an oil extracted from domestic fowl eggs, and particularly the yolks, obtained by feeding egg-laying domestic fowls with xcfx896 highly unsaturated fatty acid and optionally xcfx893 highly unsaturated fatty acid, to the production process or finished formula such as dietary formula to premature infants, dietary formula to infants, a follow-up formula or milk product for expectant nursing mothers.
The types of foods to which is added lipid having a high content of arachidonic acid and optionally docosahexaenoic acid extracted from domestic fowl eggs of the present invention are not limited to formula for premature infants, formula for nursing infants, follow-up formula or milk product for expectant or nursing mothers. One example is the addition to foods containing oils, examples of which include natural foods containing oils of meat, fish and nuts, foods to which oils are added during preparation such as Chinese food, Chinese noodles and soup, foods using oil as a heat medium such as tempura, deep-fried fish, deep-fried bean curd, fried rice, doughnuts and deep-fried confections, oily foods or processed foods to which oils are added during processing such as butter, margarine, mayonnaise, salad dressing, chocolate, instant Chinese noodles, caramel, cookies and ice cream, as well as foods that are sprayed or coated with oil during final processing such as crackers, hard biscuits and jam-filled bread.
However, examples are not limited to foods containing oil, but rather also include agricultural foods such as bread, noodles, rice, confections, bean curd and their processed foods, fermented foods such as rice wine and medicinal liquors, livestock foods such as sweet rice wine, vinegar, soy sauce, miso, salad dressing, yogurt, ham, bacon, sausage and mayonnaise, sea foods such as boiled fish paste, deep-fried fish and fish cake, as well as beverages such as fruit juice, soft drinks, sports drinks, alcoholic beverages and tea.